Risk markers for cardiovascular disease

Abstract

Provided herein methods for determining whether a subject, particularly a human subject, is at risk of developing, having, or experiencing a complication of cardiovascular disease, and methods of treating subjects who are identified by the current methods of being at risk for cardiovascular disease. In one embodiment, the method comprises determining levels of one or more oxidized apolipoprotien A-I related biomolecules in a bodily sample from the subject. Also, provided are kits and reagents for use in the present methods. Also provided are methods for monitoring the status of cardiovascular disease in a subject or the effects of therapeutic agents on subjects with cardiovascular disease. Such method comprising determining levels of one or more oxidized apolipoprotein A-I related molecules in bodily samples taken from the subject over time or before and after therapy.

1. A method for identifying a subject at risk of having cardiovascular disease, comprising: determining levels of one or more oxidized apolipoprotein A-1 (apoA-1)-related biomolecules in a biological sample of blood, serum, or plasma from the subject, wherein the one or more oxidized apoA-1-related biomolecules are oxidized apoA-1, oxidized apoA-1 peptide fragments derived from an oxidized apoA-1, or a combination thereof;
 wherein said oxidized apoA-1 fragments are at least five amino acids in length and comprise an oxidized amino acid; 
 wherein the oxidized apoA-1 and the oxidized apoA-1 peptide fragments are lipid-free or bound to a lipid; 
 wherein the oxidized apoA-1 and the oxidized apoA-1 from which the oxidized apoA-1 peptide fragments are derived have reduced cholesterol efflux stimulating activity as compared to un-oxidized apoA-1; and 
 wherein elevated levels of the one or more oxidized apoA-1-related biomolecules in the biological sample as compared to a control value or an internal standard indicates that the subject is at risk of having cardiovascular disease. 
 
     
     
       2. The method of  claim 1 , wherein the oxidized apoA-1 peptide fragments are 6 to 242 amino acids in length. 
     
     
       3. The method of  claim 2 , wherein the method employs a procedure or reagent for detecting one or more of the following oxidized amino acid residues: chlorotyrosine, nitrotyrosine, dityrosine, trihydroxyphenylalanine, dihydroxyphenylalanine, and tyrosine peroxide. 
     
     
       4. The method of  claim 1 , wherein the method employs a procedure for detecting: (a) oxidized apoA-1 that comprises one or more oxidized amino acid residues at positions 18, 29, 33, 166, 192, or 236 in SEQ ID NO. 1; or (b) oxidized apoA-1peptide fragments that are 6-242 amino acids in length and comprise an oxidized portion of SEQ ID NO: 1, wherein said oxidized portion comprises one or more oxidized amino acid residues at positions 18, 29, 33, 166, 192, or 236 in SEQ ID NO: 1; or a combination of (a) and (b). 
     
     
       5. The method of  claim 1 , wherein levels of the one or more oxidized apoA-1related biomolecules are compared to a control value or a range of control values based upon levels of the one or more oxidized apoA-1-related biomolecules in comparable biological samples from a control population of human subjects. 
     
     
       6. A method for identifying a subject's risk of having cardiovascular disease, comprising:
 (i) determining a first risk value by comparing levels of one or more oxidized apoA-1-related biomolecules in a bodily sample of blood, serum or plasma from the subject to a control value; 
 wherein the one or more oxidized apoA-1-related biomolecules are oxidized apoA-1, oxidized apoA-1 peptide fragments derived from an oxidized apoA-1, or combinations thereof; 
 wherein said oxidized apoA-1fragments are 6 to 242 amino acids in length and comprise an oxidized amino acid; 
 wherein the oxidized apoA-1 and the oxidized apoA-1 peptide fragment are lipid free or bound to a lipid; 
 wherein the oxidized apoA-1 and the oxidized apoA-1 from which the oxidized apoA-1 peptide fragments are derived have reduced cholesterol efflux stimulating activity as compared to un-oxidized apoA-1; and
 (ii) determining one or more additional cardiovascular risk values in the subject, wherein said one or more additional risk values are obtained by 
 a) determining the subject's blood pressure; 
 b) determining levels of low density lipoprotein, or cholesterol, or both in a biological sample from the subject; 
 c) assessing the subject's response to a stress test; 
 d) determining levels of myeloperoxidase, C-reactive protein, or both in a biological sample from the subject; and 
 e) determining the subject's atherosclerotic plaque burden, and 
 
 (iii) combining said first risk value with said one or more additional risk values to provide a final risk value that is indicative of the subject's risk of having cardiovascular disease. 
 
     
     
       7. The method of  claim 1 , wherein the oxidized apoA-1 related biomolecule comprises a modified tyrosine residue. 
     
     
       8. The method of  claim 1 , wherein the oxidized apoA-1 related biomolecule comprises a modified phenylalanine residue. 
     
     
       9. The method of  claim 1 , wherein the subject is a non-smoker. 
     
     
       10. The method of  claim 1 , wherein the subject is free of symptoms for cardiovascular disease. 
     
     
       11. The method of  claim 1 , wherein the subject is nonhyperlipidemic. 
     
     
       12. The method of  claim 1 , wherein the oxidized apoA-1-related biomolecule comprises at least one chlorotyrosine residue. 
     
     
       13. The method of  claim 1 , wherein the oxidized apoA-1 related biomolecule comprises at least one nitrotyrosine residue. 
     
     
       14. The method of  claim 1 , wherein the oxidized apoA-1 related biomolecule comprises at least one dihydroxyphenylalanine residue. 
     
     
       15. The method of  claim 1 , wherein the oxidized apoA-1 related biomolecule comprises at least one trihydroxyphenylalanine residue. 
     
     
       16. The method of  claim 1 , wherein the oxidized apoA-1 related biomolecule comprises a modified amino acid that is formed when apoA-1 is reacted with a myeloperoxidase (MPO)-generated nitrating species. 
     
     
       17. The method of  claim 1 , wherein the oxidized apoA-1 related biomolecule comprises a modified amino acid that is formed when apoA-1 is reacted with an (MPO)-generated chlorinating species. 
     
     
       18. The method of  claim 1 , wherein the oxidized apoA-1 related biomolecule comprises a modified amino acid that is formed when apoA-1 is reacted with an MPO-generated tyrosyl radical generating system. 
     
     
       19. The method of  claim 1 , wherein the oxidized apoA-1 related biomolecule comprises a modified tyrosine residue, a modified phenylalanine residue, or both. 
     
     
       20. A method for identifying a subject at risk of having cardiovascular disease, comprising: determining levels of one or more oxidized apolipoprotein A-1 (apoA-1)-related biomolecules in a biological sample of blood, serum or plasma from the subject,
 wherein the one or more oxidized apoA-1-related biomolecules comprise at least one modified amino acid that inhibits the ability of the oxidized apoA-1 related biomolecules to reduce aggregation of phospholipase C-treated LDL, wherein the one or more oxidized apoA-1 related biomolecules are selected from lipid-associated oxidized apoA-1, lipid-free oxidized apoA-1, lipid-associated oxidized apoA-1 peptide fragments, and lipid-free oxidized apoA-1 peptide fragments, and 
 wherein elevated levels of the one or more oxidized apoA-1-related biomolecules in the biological sample as compared to a control value or an internal standard indicates that the subject is at risk of having cardiovascular disease. 
 
     
     
       21. A method for identifying a subject at risk of having cardiovascular disease, comprising: determining levels of one or more oxidized apolipoprotein A-1 (apoA-1)-related biomolecules in blood, serum, or plasma from the subject, wherein the one or more oxidized apoA-1-related biomolecules comprise a modified tyrosine residue, a modified phenylalanine residue, or both, wherein the one or more oxidized apoA-1 related biomolecules are selected from lipid-associated oxidized apoA-1, lipid-free oxidized apoA-1, lipid-associated oxidized apoA-1 peptide fragments, and lipid-free oxidized apoA-1 peptide fragments, wherein the oxidized apoA-1 fragments are 6 to 242 amino acids in length and comprise an oxidized amino acid; and wherein elevated levels of the one or more apoA-1-related oxidized biomolecules in the subject's blood, serum, or plasma as compared to a control value or an internal standard indicates that the subject is at risk of having cardiovascular disease. 
     
     
       22. The method of  claim 1 , wherein at least one of the one or more oxidized apoA-1-related biomolecules is oxidized apoA-1. 
     
     
       23. A method for identifying a subject at risk of having cardiovascular disease, comprising:
 (i) determining levels of one or more oxidized apolipoprotein A-1 (apoA-1)-related biomolecules in a biological sample of blood, serum, or plasma from the subject, wherein the oxidized apoA-1-related biomolecules are oxidized apoA-1, oxidized apoA-1 peptide fragments derived from an oxidized apoA-1, or both; 
 wherein the oxidized apoA-1 fragments are 6 to 242 amino acids in length and comprise an oxidized amino acid; 
 wherein the oxidized apoA-1 and the oxidized apoA-1 peptide fragment are lipid-free or bound to a lipid and wherein the oxidized apoA-1 and the oxidized apoA-1 from which the oxidized apoA-1 peptide fragments are derived has reduced cholesterol efflux stimulating activity as compared to un-oxidized apoA-1; 
 (ii) comparing the level of the one or more oxidized apoA-1-related biomolecules in the biological sample to a control value or an internal standard; and 
 (iii) characterizing the subject's risk of having cardiovascular disease based on said comparison, wherein elevated levels of the one or more oxidized apoA-1-related biomolecules as compared to the control value or the internal standard indicates that the subject is at risk of having cardiovascular disease. 
 
     
     
       24. The method of  claim 23 , wherein the oxidized apoA-1-related biomolecule comprises a modified amino acid that is formed when apoA-1 is reacted with a myeloperoxidase (MPO)-generated nitrating species, an MPO-generated chlorinating species, an MPO-generated tyrosyl radical generating system, or a combination thereof 
     
     
       25. The method of  claim 23 , wherein the-oxidized apoA-1 comprises one or more oxidized amino acid residues at positions 18, 29, 33, 166, 192, or 236 in SEQ ID NO: 1; and the oxidized apoA-1 peptide fragments comprises an oxidized portion of SEQ ID NO: 1, wherein said oxidized portion comprises one or more oxidized amino acid residues at positions 18, 29, 33, 166, 192, or 236 in SEQ ID NO: 1. 
     
     
       26. A method for identifying a subject at risk of having cardiovascular disease, comprising:
 (i) determining levels of one or more oxidized apolipoprotein A-1 (apoA-1)-related biomolecules in a biological sample of blood, serum or plasma from the subject, 
 wherein the oxidized apoA-1-related biomolecules comprise at least one modified amino acid that inhibits the ability of the oxidized apoA-1 related biomolecule to reduce aggregation of phospholipase C-treated LDL, 
 wherein the one or more oxidized apoA-1 related biomolecules are selected from lipid-associated oxidized apoA-1, lipid-free oxidized apoA-1, lipid-associated oxidized apoA-1 peptide fragments, and lipid-free oxidized apoA-1 peptide fragments, or any combination thereof; 
 wherein said oxidized apoA-1 fragments are 6 to 242 amino acids in length and comprise an oxidized amino acid; 
 (ii) comparing the level of the one or more oxidized apoA-1-related biomolecules in the biological sample to a control value or an internal standard; and 
 (iii) characterizing the subject's risk of having cardiovascular disease based on said comparison; wherein elevated levels of the one or more oxidized apoA-1-related biomolecules as compared to the control value or the internal standard indicates that the subject is at risk of having cardiovascular disease. 
 
     
     
       27. The method of  claim 26 , wherein the oxidized apoA-1-related biomolecules comprise a modified amino acid that is formed when apoA-1 is reacted with a myeloperoxidase (MPO)-generated nitrating species, an MPO-generated chlorinating species, an MPO-generated tyrosyl radical generating system, or any combination thereof. 
     
     
       28. The method of  claim 26 , wherein the oxidized apoA-1 comprises one or more oxidized amino acid residues at positions 18, 29, 33, 166, 192, or 236 in SEQ ID NO: 1; and the oxidized apoA-1 peptide fragments comprise an oxidized portion of SEQ ID NO: 1, wherein said oxidized portion comprises one or more oxidized amino acid residues at positions 18, 29, 33, 166, 192, or 236 in SEQ ID NO: 1. 
     
     
       29. The method of  claim 23 , wherein the one or more oxidized apoA-1 related biomolecules comprise a modified tyrosine residue, a modified phenylalanine residue, or both. 
     
     
       30. The method of  claim 23 , wherein the cardiovascular disease is atherosclerosis. 
     
     
       31. The method of  claim 23 , wherein the subject is a non-smoker, is free of symptoms for cardiovascular disease, and/or is nonhyperlipidemic. 
     
     
       32. The method of  claim 23 , wherein levels of the one or more oxidized apoA-1-related biomolecules are determined by an immunological technique, mass spectrometry, high performance liquid chromatography (HPLC), or a combination thereof. 
     
     
       33. The method of  claim 23 , wherein the levels of the one or more oxidized apoA-1-related biomolecules is determined using an antibody that is immunoreactive with said one or more oxidized apoA-1-related biomolecule. 
     
     
       34. The method of  claim 26 , wherein the one or more oxidized apoA-1 related biomolecules comprise a modified tyrosine residue, a modified phenylalanine residue, or both. 
     
     
       35. The method of  claim 26 , wherein the cardiovascular disease is atherosclerosis. 
     
     
       36. The method of  claim 26 , wherein the subject is a non-smoker, is free of symptoms for cardiovascular disease, and/or is nonhyperlipidemic. 
     
     
       37. The method of  claim 26 , wherein levels of the one or more oxidized apoA-1-related biomolecules are determined by an immunological technique, mass spectrometry, high performance liquid chromatography (HPLC), or a combination thereof. 
     
     
       38. The method of  claim 26 , wherein levels of the one or more oxidized apoA-1-related biomolecules is determined using an antibody that is immunoreactive with said one or more oxidized apoA-1-related biomolecules. 
     
     
       39. A method for identifying a subject at risk of having cardiovascular disease, comprising: determining levels of one or more oxidized apolipoprotein A-1 (apoA-1)-related biomolecules in a biological sample of blood, serum, or plasma from the subject, wherein the one or more oxidized apoA-1-related biomolecules are oxidized apoA-1, oxidized apoA-1 peptide fragments derived from an oxidized apoA-1, or a combination thereof;
 wherein said oxidized apoA-1 fragments are at least 7 amino acids in length and comprise an oxidized amino acid; 
 wherein the oxidized apoA-1 and the oxidized apoA-1 peptide fragments are lipid-free or bound to a lipid; 
 wherein the oxidized apoA-1 and the oxidized apoA-1 from which the oxidized apoA-1 peptide fragments are derived have reduced cholesterol efflux stimulating activity as compared to un-oxidized apoA-1; and 
 wherein elevated levels of the one or more oxidized apoA-1-related biomolecules in the biological sample as compared to a control value or an internal standard indicates that the subject is at risk of having cardiovascular disease. 
 
     
     
       40. The method of  claim 39 , wherein the method employs a procedure for detecting: (a) oxidized apoA-1that comprises one or more oxidized amino acid residues at positions 18, 29, 33, 166, 192, or 236 in SEQ ID NO. 1; or (b) oxidized apoA-1 peptide fragments that are at least 7 amino acids in length and comprise an oxidized portion of SEQ ID NO: 1, wherein said oxidized portion comprises one or more oxidized amino acid residues at positions 18, 29, 33, 166, 192, or 236 in SEQ ID NO: 1; or a combination of (a) and (b). 
     
     
       41. The method of  claim 39 , wherein levels of the one or more oxidized apoA-1 related biomolecules are compared to a control value or a range of control values based upon levels of the one or more oxidized apoA-1-related biomolecules in comparable biological samples from a control population of human subjects. 
     
     
       42. The method of  claim 39 , wherein the oxidized apoA-1 related biomolecule comprises a modified amino acid that is formed when apoA-1 is reacted with a myeloperoxidase (MPO)-generated nitrating species. 
     
     
       43. The method of  claim 39 , wherein the oxidized apoA-1 related biomolecule comprises a modified amino acid that is formed when apoA-1 is reacted with an (MPO)-generated chlorinating species. 
     
     
       44. The method of  claim 39 , wherein the oxidized apoA-1 related biomolecule comprises a modified amino acid that is formed when apoA-1 is reacted with an-MPO-generated tyrosyl radical generating system. 
     
     
       45. The method of  claim 39 , wherein the oxidized apoA-1 related biomolecule comprises a modified tyrosine residue, a modified phenylalanine residue, or both.